Project Summary/Abstract This proposal aims for proof-of-concept of vitamin B3 as an MRI probe to image NAD (i.e. Nicotinamide adenine dinucleotide) and its metabolic transformations in vivo. NAD is one of the most important coenzymes in the cell. NAD plays vital roles in energy metabolism.. NAD is also a key signaling molecule in cell homeostasis, lifespan regulation, DNA repair and telomere maintenance. Recently, there has been a considerable interest in NAD as new studies have revealed age-associated systemic NAD decline. In addition, studies have linked NAD biosynthesis to pathophysiological mechanisms in several diseases, such as diabetes, non-alcoholic fatty liver disease, retinal degeneration, multiple sclerosis, Alzheimer?s disease, or cancer. However, current ability to image NAD metabolism in vivo is severely lacking. It is either limited by penetration depth (i.e. optical imaging) or relies on radioactive tracer uptakes without providing direct information about metabolic transformations (i.e. PET/SPECT). MRI suffers from limited signal to noise ratio. In mammals, NAD is synthesized from vitamin B3 precursors. We have dreamed of labeling vitamin B3 with MRI-visible stable isotopes, enhancing the isotope signals by a million-fold by hyperpolarization techniques, injecting, and imaging the metabolic conversions of hyperpolarized vitamin B3 to NAD in vivo on an MRI scanner. Recent advances in hyperpolarized metabolic MR imaging, pioneered by the PI and others, allow tracking metabolic conversions of hyperpolarized 13C-pyruvate to downstream metabolic products, resulting in new imaging biomarkers that inform disease progression and guide treatment decisions. Similar techniques may be used to develop hyperpolarized vitamin B3 for NAD imaging but to the best of our knowledge, this novel concept has never been realized because it is unclear whether vitamin B3 has the right properties. The goal of this proof-of-concept proposal is to investigate the biochemistry and MR properties of stable isotope-labeled vitamin B3 in ex vivo organs by using Mass Spectrometry (MS) and non- hyperpolarized MR spectroscopy. We will obtain important information from the ex vivo data to help us assess the likelihood of observing NAD as a downstream metabolic product of the injected vitamin B3 within a few minutes of the hyperpolarization lifetime and estimate the signal to noise ratio of NAD in important organs if labeled vitamin B3 was to be hyperpolarized and injected. The proposed research will be guided by our extensive experience in radiotracer synthesis, MS analysis, MR spectroscopy, and the development of new hyperpolarized probes. The success of the proposed study will mark a critical milestone for the future development of vitamin B3 as a hyperpolarized probe for metabolic imaging of NAD.